Abstract
Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.
Highlights
Apoptosis, an event that is both morphologically distinguishable from other types of cell death and tightly regulated from a genetic and biochemical point of view, controls tissue homeostasis and eliminates damaged cells in mammals [1]
Whereas Cc cDNA was cloned into the C-end fragment of the yellow fluorescent protein (cYFP) vector, the cDNAs of the Cc targets were cloned into the N-end fragment of the YFP (nYFP) vector
As discussed by Hu et al [33], pBiFC-bJunYN155 and pBiFC-bFosYC155 were employed as positive controls, while pBiFC-bJunYN155 and pBiFCbFosΔZipYC155 were used as negative controls
Summary
An event that is both morphologically distinguishable from other types of cell death (e.g., senescence or necrosis) and tightly regulated from a genetic and biochemical point of view, controls tissue homeostasis and eliminates damaged cells in mammals [1]. The main processes characterizing apoptosis are driven by a cascade of proteolytic events mediated by caspases (cysteine-dependent aspartatespecific proteases), a subfamily of cysteine proteases [3]. Two different pathways – extrinsic, or death receptor-initiated [4], and intrinsic, or mitochondrial [5], pathways – are involved in the activation of these proteolytic events. DNA damage, oxidative stress and growth factor deprivation are wellknown apoptosis inducers that activate the intrinsic pathway [6], involving the permeabilization of the outer mitochondrial membrane. This event occurs prior to the release of pro-apoptotic factors (e.g., AIF, cytochrome c [Cc], Smac/DIABLO and HtrA2/Omi) from the mitochondrial intermembrane space into the cytoplasm
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